Methods and platforms for sustainable high yield terpenoid production

ABSTRACT

Transgenic plants and methods for terpenoid production leveraging such transgenic plants are provided. Such transgenic plants may comprise a first heterologous nucleic acid encoding a polypeptide having 3-hydroxy-3-methylglutarylCoA reductase activity and a second heterologous nucleic acid encoding a polypeptide that introduces de novo formation of isopentenyl phosphate in the plant. Such de novo IP production may be achieved through the overexpression of phosphomevalonate decarboxylase in conjunction with 3-hydroxy-3-methylglutarylCoA reductase, which can result in up to a 130-fold increase of terpenoid production as compared to a wild-type plant.

BACKGROUND

Terpenoids, also referred to as isoprenoids, are one of the largest andmost chemically diverse classes of primary and secondary metabolites innature. All living organisms produce terpenoids and these compoundsserve a broad range of physiological functions, including key roles inrespiration, photosynthesis, modulating membrane fluidity, reproduction,regulating growth and development, defense, and environmental sensingand adaption. Some compounds that require targeting to membranes fortheir functions (like quinones, chlorophylls, and certain proteins) areanchored by terpenoid structures. In addition to their vital biologicalroles, terpenoids are also widely used—and highly valued—in a range ofcommercially useful products such as solvents, adhesives, coatings,synthetic intermediaries, flavorings and fragrances, biofuels,nutritional supplements, insecticides and pharmaceuticals.

Despite their structural diversity, all terpenoids begin with twouniversal five-carbon isoprene-like building blocks: isopentenyldiphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Both IPP andDMAPP are derived from two independent routes—the mevalonic acid (MVA)and methylerythritol phosphate (MEP) pathways.

Interestingly, these pathways are not systematically distributed amongthe three domains of life (eukaryotes, archaea, and bacteria). Forexample, in plants, while the MEP pathway is exclusively localized inplastids, the MVA pathway is distributed between cytoplasm, endoplasmicreticulum, and peroxisomes. However, despite the two beingcompartmentally separated in the cell, metabolic cross-talk betweenthese two pathways occurs via the exchange of IPP—and, to a lesserextent, DMAPP—in both directions.

IPP and DMAPP are subsequently used in multiple compartments byshort-chain prenyltransferases to produce prenyl diphosphateintermediates, including geranyl diphosphate (GPP, C₁₀), farnesyldiphosphate (FPP, C₁₅), and geranylgeranyl diphosphate (GGPP, C₂₀).Whereas GPP synthases localize exclusively in plastids and provideprecursors for monoterpenes, FPP synthases (FPPS) localize in cytosoland mitochondria and produce FPP for sesquiterpene, homoterpene,triterpene, sterol, brassinosteroid, and polyprenol biosynthesis. GGPPsynthases reside in plastids, mitochondria, and the endoplasmicreticulum, and produce precursors for gibberellins, homoterpenes,carotenoids, phytyl side-chains for chlorophyll/tocopherols/quinones,polyprenols, oligoprenols, abscisic acid, and strigolactones, amongothers. Recently the present inventors have shown that there is acytosolic pool of isopentenyl phosphate (IP), which can bephosphorylated to IPP and serves as a metabolically available carbonsource for production of both mono- and sesquiterpenes.

In spite of the economic significance of the terpenoids and their manyessential functions, there are many open questions about terpenoidmetabolism and its regulation in plants. There are likely sophisticatedbiological control mechanisms in place that regulate the plant'sproduction of these often structurally complex compounds. Furthermore,the origin of IP and possibly dimethylallyl phosphate (DMAP) in plantsis unresolved (i.e. the precursors for IPP and DMAPP), leaving openquestions about how the metabolism of the universal five-carbonterpenoid building blocks, IPP and DMAPP, are regulated in the plantkingdom.

Due to the complexities of terpenoid metabolism in plants, thecapabilities of microbes producing terpenoids has been investigated.While some progress has been achieved in engineering terpenoidbiosynthesis in microbes, this approach suffers from several drawbacksincluding dependence on exogenous carbon feedstocks, the toxicity ofsome terpenoids to their heterologous host, and the availability ofoften incomplete elucidated biosynthetic pathways for target productformation. In contrast, metabolic engineering in plants overcomes manyof these issues using sustainable photosynthetic carbon fixation,complementation of unknown biosynthetic steps within terpenoidbiosynthetic networks by endogenous plant enzymes, and sequestrationand/or storage of terpenoids in specialized plant tissues andstructures. Moreover, engineering of terpenoid production in crops oftenenhances natural plant defenses, thus serving as alternative pest and/orpathogen-management strategies. However, conventional approaches onlymanipulate the plastidial MEP pathway to increase terpenoid productionin plants and have, at best, only seen a modest increase in terpenoidproduction (e.g., at best, a 2-fold increase).

BRIEF SUMMARY

Transgenic plants are provided that overexpress at least twoheterologous nucleic acids that significantly increase terpenoidbiosynthesis in such transgenic plant. In at least one exemplaryembodiment, a transgenic plant is provided that comprises a firstheterologous nucleic acid encoding a polypeptide having3-hydroxy-3-methylglutarylCoA reductase (HMGR) activity and (1) a secondheterologous nucleic acid encoding a polypeptide that introduces de novoformation of isopentenyl phosphate (IP) in the transgenic plant, or (2)a polypeptide having phosphomevalonate kinase (PMK) activity. Thepolypeptide having HMGR activity and the polypeptide encoded by thesecond heterologous nucleic acid are overexpressed in the transgenicplant as compared to a corresponding wild-type plant.

In at least one embodiment, the first heterologous nucleic acid encodinga polypeptide having HMGR activity may be from Arabidopsis. One or bothof the first and second heterologous nucleic acids may optionally beoperably linked to a regulatory element for directing expression of thefirst and second heterologous nucleic acids. For example, and withoutlimitation, such a regulatory element may comprise a tissue-specificpromoter for directing expression of the first or second heterologousnucleic acid in the plant cells of a leaf, root, flower, developingovule or seed of the transgenic plant. Further, the transgenic plant maybe selected from the group consisting of: tobacco, rice, flax, wheat,barley, rye, corn, potato, pea, lettuce, cabbage, cauliflower, broccoli,turnip, radish, spinach, asparagus, onion, pepper, celery, squash,pumpkin, cucumber, strawberry, grape, raspberry, blackberry, pineapple,avocado, mango, banana, soybean, tomato, sorghum, sugarcane, algae, andany other land or water plant that is suitable for use in the disclosedsystems and methods.

Where the polypeptide encoded by the second heterologous nucleic acidintroduces de novo formation of IP in the transgenic plant and exhibitsphosphomevalonate decarboxylase (MPD) activity, the second heterologousnucleic acid may, for example and without limitation, comprise abacterial gene. There, in at least one exemplary embodiment, thetransgenic plant can produce an increased amount of metabolicallyavailable IP relative to an amount of metabolically available IPproduced in a corresponding wild-type plant due to such de novoformation of IP initiated by the second heterologous nucleic acid.

The transgenic plants of the present disclosure may also comprise athird heterologous nucleic acid comprising a sequence that encodes asynthase for catalyzing the formation of an exogenous terpenoid productof interest. There, the resulting transgenic plant expresses at least aportion of the exogenous terpenoid product of interest. In this manner,the transgenic plan can be designed to produce large amounts of aspecific terpenoid(s) (either endogenous or exogenous) as needed.

In yet another exemplary embodiment of the transgenic plants of thepresent disclosure, the polypeptide encoded by the second heterologousnucleic acid may have or comprise PMK activity. There, monoterpene andsesquiterpene production of the transgenic plant may be at or near20-fold greater and at or near 130-fold greater, respectively, thanmonoterpene and sesquiterpene production in the corresponding wild-typeplant.

Methods for producing terpenoids using at least one transgenic plant ofthe present disclosure are also provided. In at least one exemplaryembodiment, such a method for producing terpenoids using a transgenicplant comprising the steps of: providing a transgenic plant comprising afirst heterologous nucleic acid encoding a polypeptide having HMGRactivity and a second heterologous nucleic acid encoding a polypeptidehaving MPD or PMK activity such that the first and second heterologousnucleic acids are overexpressed in the transgenic plant as compared to acorresponding wild-type plant; and growing the transgenic plant underdesired conditions such that one or more terpenoids of interest areproduced. For example, in at least one embodiment, the transgenic plantis grown in the presence of labeled carbon dioxide, water, or acombination thereof; however, it will be understood that the transgenicplants may be grown under any suitable conditions whether now known orhereinafter developed.

Optionally, such methods may further comprise the step of isolating oneor more terpenoids of interest from the transgenic plant after growth.

Transgenic plants resulting from the methods described herein canproduce one or more terpenoids of interest at or over a 20-fold increaserelative to terpenoid production of each terpenoid of interest in acorresponding wild-type plant.

In at least one embodiment, the step of providing a transgenic plant mayfurther comprise the steps of: transforming plant cells withAgrobacterium containing a vector carrying the first heterologousnucleic acid encoding a polypeptide having HMGR activity in a contextthat allows for the overexpression of the first heterologous nucleicacid in a transgenic plant; transforming the plant cells with a genecontaining a vector carrying the second heterologous nucleic acidencoding a polypeptide having PMK activity in a context that allows forthe overexpression of the second heterologous nucleic acid in atransgenic plant; selecting transformants that overexpress both thefirst and second heterologous nucleic acids; and growing thetransformants into a transgenic plant.

Additionally or alternatively, the methods of the present disclosure maycomprise the step of transforming plant cells with a nucleic acidsequence operably linked to one or more regulatory elements fordirecting expression of the nucleic acid sequence in the plant cells,the nucleic acid sequence encoding a synthase for catalyzing theformation an exogenous terpenoid product of interest. Further, the stepof selecting transformants that contain and overexpress both the firstand second heterologous nucleic acids may further comprise selectingtransformants that express at least a portion of the exogenous terpenoidproduct of interest.

Other embodiments provide methods for producing a transgenic plant cellculture. In at least one embodiment, such a method may comprise thesteps of: obtaining transgenic plant cells by co-transforming plantcells with: a first heterologous nucleic acid encoding a polypeptidehaving HMGR activity and a second heterologous nucleic acid encoding apolypeptide having PMK activity; culturing a plurality of the transgenicplant cells; and selecting and isolating from the plurality oftransgenic plant cells a subset of transgenic plant cells whereexpression of the first and second nucleic acids are amplified ascompared to wild-type resulting in a transgenic plant cell culture.Furthermore, the method steps may additionally comprise the step ofgrowing transgenic plant tissue from the subset of transgenic plantcells. In certain embodiments, the resulting transgenic plant cellculture produces β-caryophyllene and 5-epi-aristolochene at or near a17-fold and a 63-fold increase, respectively, relative toβ-caryophyllene and 5-epi-aristolochene emission in a correspondingwild-type plant cell culture.

In at least one embodiment, the step of obtaining transgenic plant cellsmay further comprise co-transforming the plant cells with a thirdnucleic acid comprising a sequence encoding a synthase for catalyzingthe formation an exogenous terpenoid product of interest. Furthermore,the method may comprise the step of cultivating the transgenic plantcells so that at least a portion of the exogenous terpenoid product ofinterest encoded by the third nucleic acid is expressed by thetransgenic cells. There, the subset of transgenic plant cells in theselecting and isolating step may also further comprise transgenic plantcells expressing at least a portion of the exogenous terpenoid productof interest.

BRIEF DESCRIPTION OF THE DRAWINGS

The disclosed embodiments and other features, advantages, and aspectscontained herein, and the matter of attaining them, will become apparentin light of the following detailed description of various exemplaryembodiments of the present disclosure. Such detailed description will bebetter understood when taken in conjunction with the accompanyingdrawings, wherein:

FIGS. 1A and 1B are graphical schematics of the terpenoid biosyntheticpathways in plants and in some archaea and bacteria;

FIG. 2A shows a bar graph depicting the results of BIOMOL Greenend-point assays detecting free phosphate, where the assays wereconducted with 0.1 μM purified Nudx enzyme, 0.1 mM IPP, 10 mM MgCl₂, and0.1 M TAPS pH 8.5 at 37° C., quenched after one hour by the addition ofBIOMOL Green and phosphate detected at 623 nm; data presented are meansof independent biological experiments where n=8 for AtNudx1, AtNudx3,AtNudx6, AtNudx7, AtNudx9, AtNudx11, AtNudx12, AtNudx15, AtNudx20,AtNudx23, AtNudx24, AtNudx25, AtNudx26, and AtNudx27;

FIG. 2B shows a results of assay optimization for A. thaliana Nudx1(subpart1 and subpart 2) and Nudx3 (subpart 3 and subpart 4), withsubparts 1 and 3 representative of pH optimum and subparts 2 and 4representative of Mg²⁺ dependence and where the assays were conducted at37° C. with 0.1 mM IPP and 50 nM AtNudx1 or 25 nM AtNudx3, no activitywas observed with CaCl₂) and ZnCl₂ for both enzymes and data aremeans±SD (n=3 independent experiments);

FIGS. 3A-3C show crystal structures of active site electron density mapsof the following at pH 5.0: (1) IPP- and Mg²⁺-bound AtNudx1 (FIG. 3A),showing unbiased Fo-Fc electron density omit map at contour level 2σ forIPP, active site Mg²⁺ and coordinating waters 302; (2) Mg²⁺ andphosphate coordination scheme in wild-type AtNudx1-IPP (FIG. 3B), withside chains of key residues shown as well; and (3) GPP-bound E56A mutantof AtNudx1 (pdb 5GP0) (FIG. 3C), with side chains of key residues shownin FIG. 3B shown in FIG. 3C as well for comparison;

FIG. 4 demonstrates substrate recognition by A. thaliana Nudx1 and E.coli and human 8-oxo-dGTPase as displayed using crystal structures, withsubpart a of FIG. 4 showing AtNudx1 with IPP bound (residue sidechainslining the active site pocket shown and Mg²⁺ and phosphate interactingresidues omitted for clarity), subpart b of FIG. 4 showing E. coli8-oxo-dGTPase from with 8-oxo-dGMP (80G) bound (pdb 3A6T), and subpart cof FIG. 4 showing human 8-oxo-dGTPase with 8-oxo-dGMP bound (pdb 3ZR0)(side chains of key residues involved in the nucleotide binding shown);

FIG. 5A shows a bar graph representative of AtNudx1 and AtNudx3expression in various Arabidopsis tissues; FIG. 5B shows a bar graph ofthe AtNudx1 and AtNudx3 transcript level data from Col-O and T-DNAinsertion mutants determined by qRT-PCR; FIGS. 5C-5E show bar graphsrepresentative of the effect of AtNudx1 and AtNudx3 knockouts on steroland terpenoid formation, with β-Caryophyllene (FIG. 5C) and linalool(FIG. 5E) emission measured from flowers of 5-week-old Arabidopsisinflorescences, and sterol levels (FIG. 5D) measured in 8-day-oldArabidopsis seedlings of Col-O and nudx1 and nudx3 mutants; and FIG. 5Fshows a bar graph displaying data relative to the transientoverexpression of AtNudx1 and AtNudx3 under control of a CaMV-35Spromoter in tobacco leaves according to at least one embodiment of thepresent disclosure; wherein, all data displayed in FIGS. 5A-5F aremeans±s.e.m., n=3 biological independent samples, except (1) n=6biological independent samples for Col-O in the data shown in FIGS. 5Cand 5E, (2) *P<0.05; **P 0.01; ****P=0.0001 (two-tailed Student'st-test); (3) FW, fresh weight; and (4) nd, not detected;

FIG. 6, subparts a-r, show tissue-specific expression of AtNudx1 andAtNudx3 (prepared using gene-type promoters fused to β-glucuronidase(GUS) reporter, the expression of which was verified using GUS stainingsolution), with AtNudx1 promoter-GUS shown in subparts a, c, e, g, i, k,m, o, and q, and AtNudx3 promoter-GUS shown in subparts b, d, f, h, j,1, n, p, and r, and reporter gene expression patterns in mature flowers(subparts a and b), petals (subparts c and d), anthers (subparts e andf), stigmas and styles (subparts g and h), whole rosettes (subparts iand j), roots (subparts k and 1), guard cells (subparts m and n),trichomes (subparts o and p), and 7-day old seedlings (subparts q and r)(scale bars: 100 μm for subparts e, f, g, h, k, l, m, n, o, and p); 500μm for subparts a, b, c, d, q, and r; and 3 cm for subparts i and j);

FIG. 7A shows the structure of the AtNudx1 green with two exonspresented as filled and open 5′ and 3′ UTR boxes, with the arrowsshowing the positions of forward and reverse primers used for qRT-PCRanalysis;

FIG. 7B shows the structure of the AtNudx3 gene with 21 exons presentedas filled and opens 5′ and 3′ UTR boxes, with the arrows showing thepositions of forward and reverse primers used for qRT-PCR analysis;

FIG. 8 shows a bar graph representative of levels of expression ofAtNudx1 and AtNudx3 mRNAs in wild-type tobacco leaves that wereinfiltrated with agrobacterium carrying an empty vector control (EV), anAtNudx1 construct and an AtNudx3 construct, where absolute transcriptlevels of AtNudx1 (white bar) and AtNudx3 (gray bar) are shown as pg/200ng total RNA (means±s.e.m., n=3 biologically independent samples);

FIGS. 9A-9C display graphical data relating to the effect of Roseiflexuscastenholzii MPD overexpression on terpenoid formation in tobacco, withFIG. 9A representative of the RcMPD transcript levels, determined byqRT-PCR, in wild-type and transgenic tobacco lines prepared pursuant tothe present disclosure (empty vector control EV, MPD-1, MPD-4, andMPD-11), FIG. 9B representative of sterol levels in tobacco leaves ofwild-type, EV control-, and RcMPD-overexpressing lines prepared pursuantto the current disclosure (data are means±s.e.m., n=5 biologicallyindependent samples for WT and MPD-11, n=3 biological independentsamples for EV, and n=6 biologically independent samples for MPD-1 andMPD-4), and FIG. 9C representative of emission of monoterpenes,β-caryophyllene and 5-epi-aristolochene, from tobacco leaves ofwild-type, EV control- and RcMPD-overexpressing lines prepared pursuantto the present disclosure (data are means±s.e.m., n=6 biologicallyindependent samples for WT, except n=3 biologically independent samplesfor EV and MPD-11, n=5 biologically independent samples for MPD-1, andn=8 biologically independent samples for and MPD-4); *P<0.05; **P<0.01;***P=0.001 (two-tailed Student's t-test); nd=not detected;

FIGS. 10A-10E display graphical data from the analysis ofterpenoid-quinone conjugates in tobacco leaves of wild-type, EV control,and RcMPD overexpressing lines, with FIG. 10A representative of theamount of the MVA 100-derived terpenoid-quinone conjugate, ubiquinone ineach sample and FIGS. 10B-10E representative of the amounts ofMEP-pathway 102 derived terpenoid-quinone conjugates in each sampleincluding plastoquinone (FIG. 10B), α- and γ-tocopherols (FIGS. 10C and10D, respectively), and phylloquinone (FIG. 10E) (data are means±s.e.m.,n=4 biologically independent samples, except for EV in FIG. 10A andRcMPD-11 in FIG. 10D, which are n=3);

FIGS. 11A and 11B display graphical data representative of the effect ofoverexpression of AtIPK and AtHMGR1 on terpenoid formation in a RcMPD-4tobacco transgenic line of the present disclosure, where FIG. 11A showsa bar graph of levels of AtIPK and AtHMGR1 mRNAs in tobacco leaves ofthe RcMPD-4 transgenic line infiltrated with Agrobacterium carrying theempty vector control (MPD-EV) (black bars), the AtIPK construct (whitebars), and the AtHMGR1 construct of the present disclosure (gray bars)and absolute transcript levels of AtIPK and AtHMGR1 are shown aspicograms per 200 ng total RNA (means±s.e.m., n=3 biologicallyindependent samples), and FIG. 11B shows a bar graph of sesquiterpeneand monoterpene emission from RcMPD-4 transgenic tobacco leavestransiently overexpressing EV, AtIPK, and AtHMGR1, with all datameans±s.e.m. (n=3 biologically independent samples); *P<0.05; **P<0.01;nd=not detected;

FIG. 12A displays data relating to levels of SaSSy and AtHMGR1 mRNAs inwild type and RcMPD-4 transgenic tobacco leaves infiltrated withAgrobacterium carrying SaSSy construct alone and AtHMGR1 construct withthe SaSSy construct (absolute transcript levels of SaSSy and AtHMGR1 areshown as pg/mg total RNA (means±s.e.m., n=3 biologically independentsamples);

FIGS. 12B and 12C display data relating to the emission of introducedsantalenes (α-exo-bergamotene and α-santalene), with wild type and MPD-4tobacco leaves used as genetic background for transient overexpressionof AtHMGR1 and SaSSy in different combinations and shown on the y-axis(data are means±SEM, n=3 biologically independent samples; *P<0.05;**P<0.01 (two-tailed Student's t-test);

FIG. 13 illustrates, for each GFP fusion construct a-f, a schematicdiagram on the left and the corresponding transient expression in N.benthiamiana leaves detected by confocal laser scanning microscopy shownon the right, with construct a: a transient overexpression of GFP emptyvector control, construct b: a RcMPD fused to an N-terminal of GFP(MPD-GFP), construct c: RcMPD fused to a C-terminal GFP (GFP-MPD), withall GFP fluorescence and chlorophyll autofluorescence in constructs a-cshown in the left and middle panels, respectively, while the mergedpanels show the overlay of GFP and chlorophyll fluorescence (GFP aloneand chlorophyll autofluorescence were used as cytosolic and plastidicmarkers, respectively); and constructs d-f showing coinfiltration ofRcMPD GFP constructs with peroxisomal (px-rk) (constructs d and e) andmitochondrial (mt-rk) (construct e) markers, labeled with RFP and shownin the middle, with the merged panels showing the overlay of GFP and RFPfluorescence (these experiments were repeated independently three timeswith similar results; scale bar, 5 μm);

FIG. 13g shows the sequence alignment of RcMPD with Arabidopsis AtMDD1and AtMDD2. The identified putative PTS2 motifs AtMDD1 and AtMDD2 arelabelled in box 1301. The traditional plant PTS2 signal, (R/K) (L/V/I)X5 (H/Q) (L/A) was identified in RcMPD and is labeled in box 1302;

FIGS. 14A and 14B show graphical data regarding the effect of AtPMK andAtHMGR1 overexpression on terpenoid formation in transgenic tobaccoleaves produced pursuant to the current disclosure, where FIG. 14A showslevels of AtPMK and AtHMGR1 mRNAs in wild-type tobacco leavesinfiltrated with Agrobacterium carrying the empty vector control (WT-EV)(black bars), the AtPMK construct (white bars), the AtHMGR1 construct(gray bars) and the AtPMK construct with the AtHMGR1 construct (dottedbars) (absolute transcript levels of AtPMK and AtHMGR1 are shown aspicograms per 200 ng total RNA (means±s.e.m., n=3 biologicallyindependent samples)); and FIG. 14B shows sesquiterpene and monoterpeneemission from tobacco wild-type leaves transiently overexpressing EV,AtPMK, AtHMGR1, and AtPMK and AtHMGR1 together (all data aremeans±s.e.m., n=3 biologically independent samples, except n=6biologically independent samples for WT-EV; *P<0.05; **P<0.01;***P<0.001 (two-tailed Student's t-test); and

FIG. 15 shows a flow-chart representative of method 1500 for producingterpenoids using the transgenic plant and/or cells of the presentdisclosure.

While the present disclosure is susceptible to various modifications andalternative forms, exemplary embodiments thereof are shown by way ofexample in the drawings and are herein described in detail.

DETAILED DESCRIPTION

For the purposes of promoting an understanding of the principles of thepresent disclosure, reference will now be made to the embodimentsillustrated in the drawings and specific language will be used todescribe the same. It will nevertheless be understood that no limitationof scope is intended by the description of these embodiments. On thecontrary, this disclosure is intended to cover alternatives,modifications, and equivalents as may be included within the spirit andscope of this application as defined by the appended claims. Aspreviously noted, while this technology may be illustrated and describedin one or more preferred embodiments, the compositions, systems andmethods hereof may comprise many different configurations, forms,materials, and accessories.

In the following description, numerous specific details are set forth inorder to provide a thorough understanding of the present disclosure.Particular examples may be implemented without some or all of thesespecific details and it is to be understood that this disclosure is notlimited to particular biological systems, which can, of course, vary.

Various techniques and mechanisms of the present disclosure willsometimes describe a connection or link between two components. Wordssuch as attached, linked, coupled, connected, and similar terms withtheir inflectional morphemes are used interchangeably, unless thedifference is noted or made otherwise clear from the context. Thesewords and expressions do not necessarily signify direct connections, butinclude connections through mediate components and devices. It should benoted that a connection between two components does not necessarily meana direct, unimpeded connection, as a variety of other components mayreside between the two components of note. Consequently, a connectiondoes not necessarily mean a direct, unimpeded connection unlessotherwise noted.

Furthermore, wherever feasible and convenient, like reference numeralsare used in the figures and the description to refer to the same or likeparts or steps. The drawings are in a simplified form and not to precisescale. It is understood that the disclosure is presented in this mannermerely for explanatory purposes and the principles and embodimentsdescribed herein may be applied to devices and/or system components thathave dimensions/configurations other than as specifically describedherein. Indeed, it is expressly contemplated that the size and shapes ofthe composition and system components of the present disclosure may betailored in furtherance of the desired application thereof.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of skill in therelevant arts. Although any methods and materials similar to orequivalent to those described herein can be used in the practice ortesting of the subject of the present application, the preferred methodsand materials are described herein. Additionally, as used in thisspecification and the appended claims, the singular forms “a”, “an” and“the” include plural referents unless the content clearly dictatesotherwise. Thus, for example, reference to “a tRNA” includes acombination of two or more tRNAs; reference to “bacteria” includesmixtures of bacteria, and the like.

Further, as used herein, the terms “gene overexpression” and“overexpression” (when used in connection with a gene) have the meaningascribed thereto by one of ordinary skill in the relevant arts, whichincludes (without limitation) the overexpression or misexpression of awild-type gene product that may cause mutant phenotypes and/or lead toabundant target protein expression.

“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides andpolymers thereof in either single- or double-stranded form, andcomplements thereof. The term encompasses nucleic acids containing knownnucleotide analogs or modified backbone residues or linkages, that aresynthetic, naturally occurring, and non-naturally occurring, havesimilar binding properties as the reference nucleic acid, andmetabolized in a manner similar to the reference nucleotides. Examplesof such analogs include, without limitation, phosphorothioates,phosphoramidates, methyl phosphonates, chiral-methyl phosphonates,2-O-methyl ribonucleotides, and peptide-nucleic acids.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues, apolypeptide, or a fragment of a polypeptide, peptide, or fusionpolypeptide. The terms apply to amino acid polymers in which one or moreamino acid residue is an artificial chemical mimetic of a correspondingnaturally occurring amino acid, as well as to naturally occurring aminoacid polymers and non-naturally occurring amino acid polymers.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the corresponding naturally occurringamino acids. Naturally occurring amino acids are those encoded by thegenetic code, as well as those amino acids that are later modified,e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Aminoacid analogs refers to compounds that have the same basic chemicalstructure as a naturally occurring amino acid, i.e. a carbon that isbound to a hydrogen, a carboxyl group, an amino group, and an R group(e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium). Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid. Amino acids may bereferred to herein by either their commonly known three letter symbolsor by the one-letter symbols recommended by the IUPAC-IUB BiochemicalNomenclature Commission. Nucleotides, likewise, may be referred to bytheir commonly accepted single-letter codes.

As used herein, the term “regulatory element” means and includes, in itsbroadest context, a polynucleotide molecule having gene regulatoryactivity, i.e. one that has the ability to affect the transcription ortranslation of an operably linked transcribable polynucleotide molecule.Indeed, regulatory elements comprise a series of nucleotides thatdetermines if, when, and at what level a particular gene is expressed.Regulatory elements such as promoters, leaders, introns andtranscription termination regions are polynucleotide molecules havinggene regulatory activity that play an integral part in the overallexpression of genes in living cells. Promoters may be derived from aclassical eukaryotic genomic gene, including (without limitation) theTATA box often used to achieve accurate transcription initiation, withor without a CCAAT box sequence and additional regulatory or controlelements (i.e. upstream activating sequences, enhancers, and silencers)or may be the transcriptional regulatory sequences of a classicalprokaryotic gene. The term “promote” may also be used herein to describea synthetic or fusion molecule, or derivative that confers, activates,or enhances expression of a nucleic acid molecule in a cell, tissue, ororgan. Promoters may contain additional copies of one or more specificregulatory elements to further enhance expression and/or to alter thespatial expression and/or temporal expression of a nucleic acidmolecule, or to confer expression of a nucleic acid molecule to specificcells or tissues such as meristems, leaves, roots, embryo, flowers,seeds or fruits (i.e. a tissue-specific promoter). In the context of thepresent invention, a promoter preferably is a plant-expressible promotersequence, meaning that the promoter sequence (including any additionalregulatory elements added thereto or contained therein) is at leastcapable of inducing, conferring, activating, or enhancing expression ina plant cell, tissue or organ. Promoters that also function or solelyfunction in non-plant cells such as bacteria, yeast cells, insect cells,and animal cells, however, are not excluded from the invention hereof.

As used herein, the term “operably linked” means a first polynucleotidemolecule, such as a promoter, connected with a second transcribablepolynucleotide molecule, such as a gene of interest, where thepolynucleotide molecules are so arranged that the first polynucleotidemolecule affects the function of the second polynucleotide molecule. Thetwo polynucleotide molecules may or may not be part of a singlecontiguous polynucleotide molecule and may or may not be adjacent. Forexample, a promoter is operably linked to a gene of interest if thepromoter modulates transcription of the gene of interest in a cell.

The terms “terpenoids” and “terpenes” are used interchangeably herein asthe inventive disclosure is generically applicable to both. Chemically,terpenes are derived from one or more isoprene unit(s), also designatedas prenyl units. By conjugation of heteroatoms such as, e.g., oxygen ornitrogen, terpenes can be modified to terpenoids that are also known asisoprenoids.

As used herein, the term “transgenic plants” refers to plants and plantcells that have incorporated DNA sequences including, but not limited togenes that are perhaps not normally present, DNA sequences not normallytranscribed into RNA or translated into a protein (“expressed”), or anyother genes or DNA sequences that one desires to introduce into thenon-transformed plant, but which one desires to either geneticallyengineer or to have altered expression. It is contemplated that in someinstances the genome of transgenic plants of the present invention willhave been augmented through the stable introduction of the transgene;however, in other instances, the introduced gene will replace anendogenous sequence. A transgenic plant includes a plant regeneratedfrom an originally-transformed plant or cell of the present disclosureand progeny transgenic plants from later generations or crosses of atransformed plant described herein.

The novel transgenic plants and methods of the present disclosure arebroadly directed toward transgenic plants, and methods of leveragingsuch inventive transgenic plants, to produce terpenoids or isoprenes inamounts significantly greater than those produced by a correspondingwild-type plant and/or as heretofore been possible using conventionalmethodologies. In at least one embodiment, the transgenic plants,platforms, and inventive methods of the present disclosure provide forat least a 20-50 fold greater increase in terpenoid production ascompared to the wild-type plant. In at least one embodiment, theinventive transgenic plants, platforms, and methodologies of the presentdisclosure increase the metabolically available isopentenyl phosphate(IP), which results in measurable changes in terpene products derivedfrom both the methylerythritol phosphate (MEP) and mevalonate (MVA)pathways. As explained in further detail below, this is achieved througha novel manipulation of the cytosolic MVA pathway in conjunction withintroducing de novo IP formation through the overexpression of one ormore bacterial genes that encode enzymes for catalyzing a rate-limitingstep of the alternate MVA pathway (e.g., bacterial phosphomevalonatedecarboxylase (MPD)).

Further, in view of the novel findings disclosed herein, previouslyunpredicted peroxisomal localization of bacterial MPD led to thediscovery that the step catalyzed by phosphomevalonate kinase (PMK) inthe classical MVA pathway imposes a hidden constraint on the fluxtherethrough. These complementary findings fundamentally alterconventional views of metabolic regulation of terpenoid metabolism inplants and provide novel metabolic engineering targets for theproduction of high-value terpenes in plants. Certain embodiments of theinventive transgenic plants, platforms, and methods provided herein alsoincrease PMK availability and, thus, increase downstream flux throughthe classical MVA pathway which can result in a significant increase inIP production and thereafter terpenoid production.

While many of the transgenic plants in the examples set forth herein aretobacco plants, because plants have a common MVA pathway, thedescription of the present disclosure is applicable to all plants andthe inventive transgenic plants, platforms and methods presented hereinmay be (or may be used in connection with) any plant or plant cell thatmay be transformed with the desired nucleic acid sequences. For example,and without limitation, the present disclosure is applicable to at leasttobacco, rice, flax, wheat, barley, rye, corn, potato, pea, lettuce,cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus,onion, pepper, celery, squash, pumpkin, cucumber, strawberry, grape,raspberry, blackberry, pineapple, avocado, mango, banana, soybean,tomato, sorghum, and sugarcane plants, and even algae (as, like landplants, algae have both the MVA and MEP pathways which act as thegatekeepers to the various branches of terpenoid biosynthesis in bothland plants and algae).

As previously described, despite their structural diversity, allterpenoids begin with two universal five-carbon isoprene-like buildingblocks, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate(DMAPP), which may be derived from two different routes: the mevalonicacid (MVA) and methylerythritol phosphate (MEP) pathways. Now referringto FIG. 1A, flow charts representative of the classical MVA pathway 100a and the MEP pathway 102 are shown. Further, FIG. 1B depicts a flowchart representative of an alternative MVA pathway 100 b that is presentin some bacteria and archaea (e.g., the Chloroflexi phylum).

The MVA pathway 100 a generates IPP and DMAPP, which are elongated bythe ubiquitous enzymes farnesyl diphosphate synthases (FPPSs).Generally, FPPSs catalyze the condensation of one DMAPP molecule withtwo IPP molecules to produce farnesyl diphosphate (FPP) and twomolecules of pyrophosphate. FPP is an essential metabolite used forsesquiterpene, homoterpene, triterpene, sterol, brassinosteroid, andpolyprenol biosynthesis. It was conventionally thought that HMGRcatalyzes the rate-limiting step of the MVA pathway within thecytoplasm; however, conventional strategies that employHMGR-overexpression-based metabolic engineering alone do not achievehigh-yield terpenoid production in plants. Instead, as supported by thepresent disclosure and data set forth herein, the MVA pathway 100 a isregulated by additional—and conventionally undetermined—mechanisms thatgovern flow through the pathway and subsequently metabolite yield.

It has recently been determined that in addition to the classical MVAand MEP pathway enzymes, plant genomes encode another IPP-generatingprotein: isopentenyl phosphate kinase (IPK). In plants, IPK localizes inthe cytoplasm, where it transforms isopentenyl phosphate (IP) andpossibly dimethylallyl phosphate (DMAP) to IPP and DMAPP viaATP-dependent phosphorylation. IPK appears to augment terpenoidproduction through both the MVA and MEP pathways 100 a, 100 b, 102.

While, in plants, IPK is involved in a metabolite reactivation process,in some bacteria and archaea, IPK catalyzes an essential and final stepin the alternative MVA pathway 100 b (see FIG. 1B). The alternative MVApathway 100 b bifurcates from the classical metabolic route 102following mevalonate kinase (MK)-mediated phosphorylation of mevalonateyielding phosphomevalonate (MVAP). In the classical MVA pathway 100 a,MVAP undergoes phosphorylation catalyzed by phosphomevalonate kinase(PMK) to produce mevalonate diphosphate (MVAPP), which is subsequentlysubjected to decarboxylation catalyzed by mevalonate 5-diphosphatedecarboxylase (MDD) (see FIG. 1A). In contrast, in the alternative MVApathway 100 b, the order of reactions is reversed. MVAP undergoesinitial decarboxylation to IP (catalyzed by a MPDD), which is followedby ATP-dependent phosphorylation of IP (catalyzed by IPK).

The presence of genes encoding IPK in all sequenced plant genomessupports that modulating the ratios of IP to IPP and DMAP to DMAPP playsa role in regulating terpenoid biosynthesis. Moreover, as described infurther detail below, a significant yield enhancement of MVA and MEPpathway-derived terpenoids was achieved upon overexpression ofArabidopsis thaliana IPK (AtIPK) in tobacco leaves, which furthersupports the contribution of IP formation to regulating the plantterpenoid network. On the other hand, the absence of genes encoding MPDsin plant genomes indicates that, in plants, IP and possibly DMAP arisevia a different route than the alternative MVA pathway 100 b.

In part, this disclosure and the data presented herein show that Nudixhydrolases function as a part of key regulatory machinery capable ofmodulating the metabolic outcome of terpenoid metabolic networks inplants. Furthermore, the data presented herein demonstrates that IP andpossibly DMAP in plants are not produced de novo, nor result from thecumulative effects of nonspecific phosphatase activity. Instead, IP andpossibly DMAP originate from the active dephosphorylation of IPP (DMAPP)by dedicated Nudix hydrolases that, together with IPK, coordinatelyregulate the concentration of the IPP destined for higher-orderterpenoid biosynthesis. These findings highlight the significance of IPKin plant metabolism and support that these Nudices, in particular Nudx3,are not just dephosphorylating isoprenoid building blocks.

The inventive transgenic plants, platforms, and methods of the presentdisclosure leverage these findings and allow for unprecedentedisoprenoid/terpenoid biosynthesis rates—in some cases over a 130-foldincrease over like terpene production in a corresponding wild-typeplant. In at least one embodiment, such transgenic plants, platforms,and methods increase metabolically available IP by overexpression of abacterial MPD, which results in a measurable uptick in terpene productsproduced (i.e. both in monoterpenes and sesquiterpenes). Moreover, asdescribed below in additional detail, the unpredicted peroxisomallocalization of bacterial MPD led to the discovery that the stepcatalyzed by PMK imposes a hidden constraint on flux through theclassical MVA pathway 100 a. Accordingly, additional embodiments of thepresent disclosure utilize the overexpression of PMK to further increasethe production of high-value terpenes by plants.

EXAMPLES

The following examples illustrate certain specific embodiments of theinvention and are not meant to limit the scope of the invention in anyway.

Example 1 Introducing De Novo IP Formation Through the Overexpression ofa Bacterial RcMPD in Tobacco

To identify IPP/DMAPP phosphatase candidates that may produce IP/DMAP inplanta, a certain unique two-domain (hydrolase/peptidase) member of theNudix hydrolase superfamily, AtNudx3 (At1g79690), was considered. Whiledata supports that AtNudx3 can dephosphorylate IPP and hydrolyzedipeptide substrates in vitro, to date, it has been difficult todetermine Nudix enzymes' physiological function due to their measurablein vitro substrate promiscuity.

Primarily, the plant material used to generate the transgenic lines andtransient expression in the studies described herein comprised N.tabacum cv. Xanthi was used for generation of transgenic lines andtransient expression because tobacco plants have the advantages of beingeasily transformed and easy to grow. However, it is noted that theresults of the studies presented herein are not limited in theirapplicability to N. tabacum as all plants have a common MVA pathway 100a. As such, it is understood that the results of these studies (as wellas the inventive transgenic plants, systems, and methodologies derivedtherefrom and presented herein) are not limited to N. tabacum, butinstead may comprise any plant or crop.

Arabidopsis T-DNA insertion mutant lines, nudx1-1 (SALK 025320C),nudx1-2 (SAIL_236_D10), nudx3-1 (SAIL_554_G07), and nudx3-2 (SALK009963), were obtained from the Arabidopsis Biological Resource Center.Homozygosity of obtained mutant lines were verified by PCR on isolatedgenomic DNA using respective gene- and T-DNA Express Primer Design (SalkInstitute). All plant material was grown in a greenhouse or growth roomunder a 16 h light/8 h dark photoperiod.

To identify putative IPP/DMAPP phosphatase candidates that produceIP/DMAP in planta, all Nudix enzymes encoded by the Arabidopsis genomein E. coli were expressed. The open reading frames (ORFs) of ArabidopsisNudix genes were PCR-amplified from cDNA with gene-specific forward andreverse primers, with the exception of AtNudx3, which was obtained fromthe Arabidopsis Biological Resource Center (clone U16680). Transitpeptide sequences were excluded when present.

ORFs were cloned into a modified pET28b vector with an N-terminalHis8-tag by the In-Fusion cloning system (Takara Bio USA). ORFs forNudix 4, 8-11, 16-18, 21-24, and 26 were also cloned into cold-shockexpression vector pCold I DNA (Takara Bio USA). The resulting constructswere transformed into BL21(DE3) E. coli and grown in terrific brothmedium supplemented with 50 μg ml⁻¹ kanamycin or 100 μg ml⁻¹ ampicillin.Protein expression was induced with 0.5 mM isopropyl1-thio-β-D-galactopyranoside at 18° C. for pET28b or 15° C. for pCold IDNA. After 20-24 hours of incubation, cells were collected bycentrifugation and lysed by sonication in 50 mM Tris-HCL pH 8.0 buffer,0.5 M NaCl, 20 mM imidazole and 10% glycerol.

After removal of the cell debris by centrifugation, expressed proteinswere purified from the supernatants by immobilized metal-affinitychromatography with HisPur Ni-NTA resin (ThermoFisher Scientific). Pureproteins were eluted with 50 mM Tris-HCL pH 8.0, 0.5 M NaCl buffersupplemented with 250 mM imidazole followed by buffer exchange to 50 mMTris-HCl pH 8.0 and 0.2 M NaCl for storage. For the crystal screeningand steady-state kinetic analysis described below, the N-terminalHis8-tags of AtNudx1 and AtNudx3 were removed with thrombin andremaining histidine-tagged protein was removed by passing over HisPurNi-NTA resin. AtNudx1 and AtNudx3 were further purified bysize-exclusion chromatography on a Superdex 200 16/60 column (GELifesciences) equilibrated and eluted with 50 mM Tris-HCl pH 8.0supplemented with 0.2 M NaCl and 2 mM dithiothreitol.

Of the 27 heterologously expressed proteins, 16 produced soluble enzymesthat were assayed for small molecule phosphatase activity (the remaininggenes, AtNudx 2, 4, 8, 10, 13, 16-19, 21, and 22 produced insolubleinclusion bodies and were not assayed). Most of these Nudix enzymes werepreviously assayed against a panel of phosphate-bearing substrates;however, isoprenoid diphosphates were notably absent.

The soluble enzymes identified were then screened for catalytic activitywith IPP as a substrate using a modified malachite green assay for freephosphate detection. As shown in the bar graph shown in FIG. 2A, onlyAtNudx1 (At1g68760) and AtNudx3 efficiently catalyzed dephosphorylationof IPP to IP, while the remaining enzymes exhibited low to no activity.Thereafter, the AtNudx1 and AtNudx3 assays were optimized for pH andmagnesium cation (Mg²⁺) dependence (see FIG. 2B), steady state kineticconstants were determined for AtNudx1 and AtNudx3 using isoprenoid mono-and di-phosphate containing compounds. Both AtNudx1 and AtNudx3catalyzed dephosphorylation of MVAPP, IPP, DMAPP, geranyl diphosphate(GPP), and FPP to the monophosphate products, MVAP, IP, DMAP, geranylphosphate (GP), and farnesyl phosphate (FP), respectively, and did notcatalyze further dephosphorylation to their respective isoprenoidalcohols. AtNudx1 utilized IPP, DMAPP, GPP, and FPP with equal catalyticefficiencies, while MVAPP was 100-fold less efficient as a substrate(Table 1). In contrast, AtNudx3 preferred IPP and DMAPP with catalyticefficiencies similar to AtNudx1 and with 3-fold higher catalyticefficiencies compared to AtNudx1 using GPP and FPP as substrates (Table1).

TABLE 1 | Kinetic parameters for recombinant A. thaliana Nudx1 andNudx3. AtNudx1 AtNudx3 K_(M) k_(cat) k_(cat)/K_(M) K_(M) k_(cat)k_(cat)/K_(M) Substrate (μm) (s⁻¹) (M⁻¹ s⁻¹) (μm) (s⁻¹) (M⁻¹ s⁻¹)(R)-MVAPP 240 ± 60 0.51 ± 0.06 2.1 × 10³ ± 0.6 125 ± 36 1.5 ± 0.2 1.2 ×10⁴ ± 0.4 DMAPP 10.7 ± 1.8  1.7 ± 0.07 1.6 × 10⁵ ± 0.3 44 ± 8 7.1 ± 0.31.6 × 10⁵ ± 0.3 IPP  8.0 ± 0.6 1.11 ± 0.02 1.4 × 10⁵ ± 0.1 36 ± 8 5.5 ±0.3 1.5 × 10⁵ ± 0.3 GPP  8.3 ± 2.5 2.0 ± 0.3 2.5 × 10⁵ ± 0.8  53 ± 132.7 ± 0.3 5.1 × 10⁴ ± 0.5 FPP  8.7 ± 1.6 1.35 ± 0.06 1.6 × 10⁵ ± 0.3  58± 10 3.3 ± 0.3 5.7 × 10⁴ ± 0.5 8-oxo-dGTP 33 ± 2 4.5 ± 0.1 1.4 × 10⁵ ±0.1 NA NA NA Data are means ± SD (n = 3 independent experiments). NA, nodetectable activity. No phosphatase activity was detected for AtNudx1and AtNudx3 with IP, DMAP, MVAP, GP, and FP.

Example 2 AtNudx1 Activity Against 8-Oxo-dGTP is Likely to be IrrelevantIn Vivo

AtNudx1 functions as an 8-oxo-dGTPase based upon homology with bacterialand mammalian Nudix superfamily members. While AtNudx3 was unable todephosphorylate 8-oxo-dGTP in the above-described studies, AtNudx1catalyzed dephosphorylation with catalytic efficiencies similar to thoseobtained with IPP and DMAPP as substrates (see Table 1). However,AtNudx1 only weakly prefers 8-oxo-dGTP as compared to dGTP with(k_(cat)/K_(M))^(8-oxo-dGTP)/(k_(cat)/K_(M))^(dGTP)=2.6²⁰, whileintracellular concentrations of dGTP are expected to be significantlyhigher than 8-oxo-dGTP.

To precisely define the structural basis for substrate selectivities ofAtNudx1, diffraction quality crystals were obtained for atomicresolution protein x-ray crystallographic analyses. Crystallizationtrials were conducted by the hanging-drop method using Hampton Researchcrystal screens. Typically, 1 μl of protein at 12 mg ml⁻¹ in 50 mMTris-HCL pH 8.0, 0.2 M NaCl and 2 mM dithiothreitol was mixed with 1 μlof each reservoir solution and incubated at 4° C. over a 500 μlreservoir solution.

Ligand-bound structures were obtained by co-crystallization with 5-10 mMligand. Diffraction-quality AtNudx1 crystals were obtained from amixture of the protein with 0.1 M succinct acid pH 5.0, 20% PEG 4000 and0.3 M Mg(NO₃)₂. Crystals were flash-frozen in cryoprotectant of 17%ethylene glycol and reservoir buffer plus substrate.

X-ray diffraction data was collected at beamlines 8.2.1 and 8.2.2 of theAdvanced Light Source at Lawrence Berkeley National Laboratory.Diffraction images were indexed and integrated with iMosflm, and themeasured reflection intensities were scaled and merged using CCP4Aimless. The initial structural elucidation of AtNudx1 was obtained bymolecular replacement with CCP4 MolRep using a search model derived fromRickettsia Felis MutT (pbd entry 4KYX) with non-conserved amino-acidresidues pruned using the CPP4 Chainsaw program. The structural modelwas refined with Phenix Refine and inspected against electron-densitymaps and adjusted manually in Coot (a model-building tool for moleculargraphics). Autobuilding was performed with Phenix Autobuild (aniterative model building, structure refinement and density modificationtool). Subsequent structure determinations of other forms of AtNudx1,which were crystalized isomorphously, were initiated with the refinedAtNudx1 model, after omission of ligands and water molecules.

TABLE 2 X-ray Diffraction Data Collection and Refinement Statistics.AtNudx1 AtNudx1 · IPP Data collection Space group P 21 21 21 P 21 21 21Cell dimensions a, b, c (Å) 36.19 73.07 116.54 36.050, 72.751, 116.390α, β. γ (°) 90.00, 90.00, 90.00 90.00, 90.00, 90.00 Resolution (Å)73.07-1.77 73.07-1.77 R_(merge) 0.109 (0.286) 0.106 (0.206) I/σI 10.7(2.4) 12.7 (5.2) Completeness (%) 88.7 (48.2) 98.6 (90 2) Redundancy 5.5(1.7) 6.4 (3.9) Refinement Resolution (Å) 61.908-2.000 61.691-1.900 No.reflections 21321 23898 R_(work)/R_(free) 0.1823/0.2165 0.1827/0.2277No. atoms Protein 2201 2196 Ligand/ion 1 35 Water 235 212 B-FactorsProtein 25.433 34.032 Ligand/ion 24.770 34.035 Water 31.275 39.342R.m.s. deviations Bond lengths (Å) 0.007 0.012 Bond angles (°) 0.8201.094

As shown in Table 2, AtNudx1 crystallized without ligands (2.0 Å,R_(work)=0.1823 and R_(free)=0.2165) and with IPP bound (1.90 Å,R_(work)=0.1827 and R_(free)=0.2277). Now referring to FIG. 3A, modelsof IPP (pdb ligand ID IPR) and three magnesium cations (Mg²⁺) are shownin active site electron density maps. IPP is clearly present in theactive site of AtNudx1 (instead of the reaction product IP), which islikely due to the low pHs (pH 5.0) and low temperatures used duringcrystallization and data collection: 4° C. and −273° C., respectively.As shown in subpart 1 of FIG. 2B, at pH 6.5 the specific activity ofAtNudx1 using IPP as a substrate is 7-fold lower than activitiesmeasured at the optimal pH of 8.5 (at 37° C.).

Like most Nudix superfamily members, AtNudx1 employs divalent cationsfor catalytic activity and, here, Mg²⁺ ions were present in thecrystallization conditions. As supported by the crystal structures shownin FIG. 3B, Mg²⁺ octahedrally coordinates several water molecules 302,the sidechains of Glu56 (E56) and Glu60 (E60), the carbonyl oxygen ofGly40 (G40), and the diphosphate oxygens of IPP. The multivalentdiphosphate group is also hydrogen bonded by His42 (H42) and Arg27(R27). As shown in subpart a of FIG. 4, the C5 carbon chain of IPP issequestered by the hydrophobic side chains of Ala11 (A11), Val12 (V12),Val13 (V13), Ile31 (I31), Ala37 (A37), Leu38 (L38), Phe78 (F78), Phe127(F127), Pro129 (P129), Leu130 (L130), and Leu133 (not labeled).

Recently, apo and GPP-bound E56A mutant structures of AtNudx1 have beenreported. The apo structures (pdb SWWD) and IPP- and GPP-boundstructures (pdb 5GP0) superimposed with root mean squared deviations of0.2969 Å and 0.3246 Å, respectively. Surprisingly, as shown in FIGS. 3Band 3C, superposition of the previously reported catalytically impairedAtNudx1 mutant (E56A) with GPP-bound (pdb 5GP0) (FIG. 3C) and theAtNudx1-IPP structure reported herein (FIG. 3B) show that the sharedchemical features of these two ligands do not superimpose. Instead, theE56A mutation likely prevents Mg²⁺ and GPP from binding in productiveconformations.

In at least one the AtNudx1-IPP structure of the present disclosureshown in FIG. 3B, Glu56 bicoordinates with two of the active site Mg²⁺ions. Mg²⁺ ions are absent in apo-AtNudx/of FIG. 3C, due to the absenceof the diphosphate group which likely initiates cation recognition andearly stages of divalent cation, active site coordination.

To compare the chemical features governing substrate binding in theseNudix enzymes, the AtNudx1-IPP complex was structurally aligned withboth 8-oxo-dGMP-bound E. coli 8-oxo-dGTPase MutT (pdb 3A6U) (see subpartb of FIG. 4) and human 8-oxo-GTPase MTH1 (pdb 3ZR0) (see subpart c ofFIG. 4). Noticeably, as shown in subpart a of FIG. 4, residues that areimportant for binding the nucleotide substrate are absent in AtNudx1(Glu34 (E34), His28 (H28), and Asn119 (N119) in MutT and Asn33 (N33),Asp119 (D119), Asp120 (D120), and Trp117 (W117) in MTH1).

Accordingly, despite sharing the same fold, AtNudx1 and 8-oxo-dGTPasespossess distinct active site pockets for substrate recognition andcatalysis. This, combined with in vitro substrate specificity studiesdescribed above, support that AtNudx1 activity with 8-oxo-dGTP is likelyinconsequential in vivo.

Example 3 AtNudx1 and AtNudx3 Contribute to IP, and Possibly DMAP,Formation in Planta

Now referring to FIGS. 5A-5F, the in planta contribution of AtNudx1 andAtNudx3 to isoprenoid production was also investigated. Primarily,AtNudx1 and AtNudx3 expression across different tissues was analyzedusing quantitative RT-PCR (qRT-PCR) with gene specific primers. Fortransient overexpression constructs, ORFs of AtNudx1 (G50379) andAtNudx3 (U16680) were obtained from the Arabidopsis Biological ResourceCenter and transferred from a Gateway-compatible entry vector into thebinary vector pB2GW7 under the control of the cauliflower mosaic virus35S promoter using the Gateway LR Clonase II (Invitrogen). Transientoverexpression was achieved by A. tumefaciens (strain EHA105 carryingthe corresponding construct) infiltration of 2-3 leaves of wild-type.Twenty-four hours after infiltration, scent emission was analyzed.

Further, RNA isolation from Arabidopsis and tobacco tissues, cDNAsynthesis and qRT-PCR analysis were performed as described in Henry etal., Orthologs of the archaeal isopentenyl phosphate kinase regulateterpenoid production in plants, Proc. Natl Acad. Sci. USA 112,10050-10055 (2015) (the “IPK Article”), the entirety of which isincorporated herein by reference. Gene-specific primers were designedusing the PrimerExpress software (Applied Biosystems). For the absolutequantification of gene transcript levels, respective cDNA fragments werepurified, diluted to several concentrations between 160 pg ml⁻¹ and 1.28pg ml⁻¹, and used to generate standards curves in qRT-PCR withgene-specific primers. Absolute quantities of the individual transcriptswere calculated on the basis of standard curves, and expressed as apictograms (pg) of mRNA per 200 ng of total RNA or as a percentage ofthe expression in wild-type. Each data point represents three biologicaland three technical replicates.

Additionally, AtNudx1 promoter-GUS and AtNudx3 promoter-GUS reportergene expression patterns were analyzed in mature flowers (each constructin 3 independent transgenic lines, all of which showed similar results,and floral and leaf volatiles collected using a closed-loop strippingsystem as is known in the art and described in the IPK Article).Approximately 2-kb regions upstream of each AtNudx1 and AtNudx3 geneswere cloned from Arabidopsis Col-0 genomic DNA with primers containingattB Gateway linkers (pNDX1_F and pNDX1_R2; pNDX3_F and pNDX3_R2). ThePCR products were inserted into the pDONR207 entry vector via a BPclonase I Gateway reaction (Invitrogen) and the fragment was sequencedto verify the identity of the insert. The insert was then moved from thepDONR207 entry vector into a pMDC163 expression vector (GUS expressionvector) by LR clonase I Gateway reaction (Invitrogen). This expressionvector was then transformed into Agrobacterium tumefaciens GV3101n andused for infiltration of A. thaliana Col-0 plants via the floral dipmethod. Hygromycin-resistant transformants were selected for GUScolorimetric assays.

Tissues were then fixed in 90% acetone for 40 minutes at −20° C., washedtwice with phosphate buffer (pH 7.0), added to GUS staining solution(0.1% Triton X-100, 10 mM EDTA, 2 mM ferricyanide, 100 mM sodium dibasicphosphate, 100 mM sodium monobasic phosphate and 4 mM5-bromo-4-chloro-3-indolyl-β-glucuronide), vacuum-infiltrated for 5minutes and incubated at 37° C. until staining was visible or for amaximum of 4 days. Tissues were then cleared in 70% ethanol for 2 daysand thereafter imaged in a 8:2:1 chloral hydrate/distilledwater/glycerol solution. The images of FIG. 6 were taken on a NikonEclipse Ti-2 inverted microscope with a Nikon Digital Sight DS-Fi2camera and expression patterns were analyzed for each construct in atleast three independent transgenic lines to account for positionaleffects of the insert.

As shown in FIG. 5A, both Nudix genes expressed in all tissues withAtNudx3 mRNA at significantly higher levels than those of AtNudx1. Inaddition, expression of a β-glucuronidase (GUS) reporter under controlof AtNudix1 and AtNudix3 promoters further indicates that theirexpression overlaps across different tissues (see FIG. 6).

To further examine the role of AtNudx1 and AtNudx3, a reverse geneticsapproach was also utilized to profile terpenoids in Arabidopsis T-DNAinsertion lines (nudx1-1, nudx1-2, nudx3-1 and nudx3-2). An AtNudx1 genewith the two exons presented as filled (coding region) and open 5′ and3′ UTR boxes (see FIG. 7A) was used. In this examination, the T-DNAinsertions (shown in gray) in the nud x1-1 and nudx1-2 mutants werelocated in exon 2 and exon 1, respectively. Further, the structure ofthe AtNudx3 gene is shown. Likewise, the AtNudx3 gene having 21 exonspresented as filled (coding region) and open 5′ and 3′ UTR boxes wasused. The T-DNA insertions, also shown in gray, in the nudx3-1 andnudx3-2 mutants were located in exon 20 and in the intron between exons14 and 15, respectively.

No AtNudx1 or AtNudx3 transcripts were detected in mutants with theexception of the nudx1-2 mutant, which, as shown in FIG. 5B, exhibited a90% reduction in AtNudx1 expression. Significantly, emission of the mostabundant sesquiterpene, β-caryophyllene, from Arabidopsis flowersincreased by 28-60% (FIG. 5C) and the concentration of the sterolsitosterol nearly doubled in all nudx mutants, while the levels ofcampesterol and stigmasterol remained unchanged (FIG. 5D). Emission ofthe monoterpene linalool from flowers was increased 148-503% in all nudxT-DNA mutants (FIG. 5E), supporting that AtNudx1 and AtNudx3 modulatethe ratios of IPP to IP and possibly DMAPP to DMAP in vivo.

Despite their differential expression levels (see FIG. 5A), the similarterpenoid metabolic profiles in the nudx1 and nudx3 mutants support thatboth AtNudx1 and AtNudx3 regulate the availability of metabolitescontributing to both GPP- and FPP-derived terpenoids. As AtNudx1 andAtNudx3 are localized in the cytoplasm, it is probable that the observedeffects on sterol levels and sesquiterpene emission result fromdephosphorylation of IPP and/or FPP (see Table 1 and FIG. 1A). Incontrast, because monoterpene formation partially relies on IPP importedinto plastids from the cytoplasm, the observed effects on monoterpenelevels can only result from IPP dephosphorylation.

Next, each Nudx gene was transiently overexpressed in wild-type N.tabacum (tobacco) leaves, which emit both monoterpene and sesquiterpenecompounds. More specifically, tobacco leaves were infiltrated withagrobacterium carrying the empty vector control (EV) or anoverexpression construct of AtNudx1 or AtNudx3 under the control of aCaMV-35S promoter (see FIG. 8 for result verification). Twenty-fourhours after infiltration, the emission of sesquiterpenes in leavesoverexpressing a Nudx gene was decreased by 57-88%, as compared toleaves infiltrated with Agrobacteria harboring an empty-vector (see FIG.5f ).

Emission of the monoterpenes linalool and β-ocimene also decreased onaverage by 50% in tobacco leaves overexpressing AtNudx1 and was lower,albeit not significantly, in tobacco leaves overexpressing AtNudx3relative to control (FIG. 5f ). Thus, overexpression of AtNudx1 andAtNudx3 resulted in an opposite metabolic phenotype to that observedwhen AtIPK was overexpressed in tobacco leaves. (see the IPK Article).When taken together, the Arabidopsis nudx1 and nudx3 mutants profiled inthe present disclosure (FIGS. 5A-5F) and the ipk mutants analyzedpreviously produce observable complimentary phenotypes, which providesin vivo evidence that Nudix and IPK catalyze opposing reactions toregulate IPP/IP and possibly DMAPP/DMAP ratios.

Thus, in plant cells, IP and DMAP formation is not the consequence ofdephosphorylation by non-specific phosphatases, but instead the resultof the catalytic activity of specific Nudix enzymes. Moreover, becauseAtNudx1 and AtNudx3 dephosphorylate FPP (Table 1), the possibility thatthese enzymes also function to modulate the FPP to FP ratio cannot beexcluded.

Accordingly, the data presented herein shows that the Nudix hydrolasesfunction as a part of the key regulatory machinery that is capable ofmodulating the metabolic outcome of terpenoid metabolic networks inplants. Furthermore, the data presented herein demonstrates that IP andpossibly DMAP in plants are not produced de novo, nor the result ofcumulative effects of nonspecific phosphatase activity. Instead, IP (andpossibly DMAP) originate from the active dephosphorylation of IPP(DMAPP) by dedicated Nudix hydrolases that regulate the concentration ofthe IPP.

Example 4 Effects of Increasing IP Formation on Terpenoid Biosynthesis

Pursuant to the above-described findings, it was determined that IP/DMAPis formed from the dedicated dephosphorylation of IPP/DMAPP catalyzed byNudix superfamily hydrolases (FIGS. 5A-5F). Considering the limitationsof the MVA pathway 100 a for high yield terpenoid production, it wasthen tested if introducing de novo IP production in plants would affectflux toward downstream terpenoids.

As plants lack genes encoding MPDs, a bacterial MPD gene fromRoseiflexus castenholzii (Rc) was used to encode an enzyme thatpossesses strict specificity for the efficient decarboxylation of MVAPto IP. To produce de novo IP, the overexpression MPD constructs wereprepared pursuant to the methodologies described in connection with theAtNudx1 and AtNudx3 studies herein, and overexpression was achieved intobacco plants as described herein or as otherwise known in the art.

The RcMPD gene was stably overexpressed in N. tabacum under control ofthe CaMV-35S promoter to create a bifurcation in the canonical MVApathway 100 and, thus, produce IP without dephosphorylating IPP. It wasthought that because the endogenous IPK is capable of convertingMPD-generated IP to IPP, then RcMPD overexpression may result in theintroduction of a novel second branch of the MVA pathway resembling thealternative MVA pathway 100 b known to exist in some bacteria andarchaea (FIG. 1B).

To generate transgenic tobacco plants overexpressing RcMPD, a RcMPD ORFwas codon-optimized for plant systems and synthesized by Clontech. Itwas subcloned using the Gateway LR Clonase II (Invitrogen) into thebinary vector pB2GW7 under the control of the cauliflower mosaic virus35S promoter. Transgenic tobacco plants were obtained via Agrobacteriumtumefaciens (strain EHA 105 carrying 35 S-RcMPD) leaf disctransformation using a standard transformation protocol. Plants rootedon BASTA selection (1 mgl-1) were screened for the RcMPD presence usingforward and reverse, RcMPD_qRT_for and RcMPD_qRT_rev, primers.Untransformed tobacco plants as well as plants transformed with emptyvector (EV) were used as controls in all experiments.

Metabolic analyses of three independent transgenic lines with differentexpression levels of the RcMPD gene, namely MPD-1, MPD-4, and MPD-11(FIG. 9A), revealed that all lines exhibited a substantial increase inoverall terpenoid production relative to wild-type and EV control plantswithout affecting expression of endogenous IPK, PMK and MDD. As shown inFIG. 9C, sterol levels, including cholesterol, stigmasterol, sitosterol,and campesterol were respectively 3.2-, 4.2-, 3.2- and 3.7-fold higherin RcMPD transgenic plants of the present disclosure relative tocontrols. (Sterol extraction and analysis were performed pursuant tomethods commonly known in the art and as described in the IPK Article.)

As shown in FIGS. 10A-10E, while more elaborate downstreamterpenoid-quinone conjugate products were not affected, this is likelydue to limited availability of the aromatic building blocks necessaryfor their biosynthesis. Finally, as shown in FIG. 9C, the leaves of MPDtransgenic plants of the present disclosure emitted up to 4.1- and7.4-fold more mono- and sesquiterpenes, respectively, than controlplants.

The increased production of terpenoids in RcMPD overexpression linesindicates that endogenous IPK can access and process de novo-produced IPto IPP and that the upstream portion of the MVA pathway 100 a providessufficient MVAP substrate to measurably increase flux through theintroduced alternative MVA pathway 100 b. In comparison to previouslygenerated AtIPK overexpression tobacco lines, the RcMPD transgenics ofthe present disclosure produced 1.5-fold more sterols and up to 2-foldhigher levels of mono- and sesquiterpenes, thus supporting that overallyields of terpenoid products from both the MVA and MEP metabolicnetworks 100, 102 and crosstalk between the two are limited byendogenous IP levels and a firm basis for leveraging these transgenicsto achieve increased terpenoid output.

To assess whether endogenous IPK in MPD transgenic plants of the presentdisclosure depleted all de novo generated IP, in at least one trialAtIPK was transiently overexpressed in the MPD-4 transgenic line(employing the heterologous expression and purification methodspreviously described, except that IPK ORFS were codon-optimized for E.coli expression and synthesized by Integrated DNA Technologies). AtIPKoverexpression in this background resulted in additional increases of1.9- and 2.8-fold in emitted sesquiterpenes β-caryophyllene and5-epi-aristolochene, respectively, relative to the levels in the MPD-4transgenic line overexpressing an empty vector (FIG. 11B).

These results support that endogenous IPK activity was limiting in theMPD transgenics. Moreover, while sesquiterpene levels increased, levelsof the emitted monoterpenes (β-ocimene and linalool) remained unchanged.This effect on the MEP-pathway 102 derived terpenoids may be caused bythe IPP transporter (involved in importing cytoplasmic IPP intoplastids, and/or plastidial enzymes acting downstream of IPP) working atmaximum capacity in these plant backgrounds and/or the occurrence of anincreased flux toward sesquiterpene formation due to the turnover of IPby IPK to relax FPP synthase inhibition, as IP and DMAP competitivelyinhibited FPP synthase.

Example 5 Overexpression of HMGR and MPD Significantly Amplifies FluxToward Downstream Products

It is conventionally thought that HMGR catalyzes the rate-limiting stepof the MVA pathway 100 a. To test whether increasing expression of HMGRin the RcMPD background further enhances terpenoid production, AtHMGR(G12571) was transiently overexpressed in RcMPD transgenic tobaccoleaves (using methods previously described). The transientoverexpression constructs were prepared as previously described inconnection with the AtNudx1 and AtNudx3 studies herein, except that forthe santalene synthase overexpression constructs, full-length SaSSy wasalso subcloned into the binary vector pB2GW7. Transient overexpressionwas achieved by A. tumefaciens (strain EHA105 carrying the correspondingconstruct) infiltration of 203 leaves of wild-type or RcMPD-4 transgenictobacco plants as described herein or as otherwise known in the art.Further, for plants co-infiltrated with multiple constructs, equalamounts of Agrobacterium cultures with OD_(600 nm) 1.0 were mixed andinfiltrated.

As supported by FIG. 11B, compared to the MPD-4 line expressing anempty-vector as a control, levels of the monoterpenes β-ocimene andlinalool increased by 2.7- and 4.6-fold, respectively, in MPD-4 linesalso overexpressing AtHMGR. Even higher levels were achieved for thesesquiterpenes β-caryophyllene and 5-epi-aristolochene, reaching 4.6-and 16.5-fold increases, respectively. When taken with the findingspreviously discussed herein, the coexpression of AtHMGR with RcMPD intobacco leaves enhances monoterpene formation by up to 20-fold andsesquiterpene production by up to or greater than 130-fold relative totheir production in wild type plants (see FIGS. 9C and 11B), which isfar greater than any production increase achieved to date usingconventional methodologies.

To further investigate the suitability of the AtHMGR-RcMPD tobaccoplatform for heterologous production of valuable terpenoids (i.e.terpenoids that are non-native to the underlying plant), Santalum albumsantalene synthase (SaSSy) was also coexpressed. FIG. 12A illustrateslevels of SaSSy and AtHMGR1 mRNAs in wild type and RcMPD-4 transgenictobacco leaves infiltrated with Agrobacterium carrying (1) the SaSSyconstruct alone, and (2) the AtHMGR1 construct in combination with theSaSSy construct. As shown in FIGS. 12A and 12B, overexpression of SaSSywith both AtHMGR and RcMPD resulted in almost 10-fold higher emittedlevels of the non-native sesquiterpenes α-exo-bergamotene andα-santalene as compared to overexpression of the SaSSy gene alone inwild type background. This data confirms that in addition to increasingproduction of terpenoids naturally produced by the underlying plantspecies, the inventive methods and platforms of the present disclosurecan be employed to increase production of terpenoids that are non-nativeto the underlying plant (i.e. not naturally occurring therein but addedthrough transformation or other techniques for achieving heterologousterpenoid production).

Example 6 PMK Contributes to Controlling Carbon Flux Through the MVAPathway

The introduced bacterial RcMPD and endogenous NtPMK genes both encodeenzymes that use MVAP as a substrate (see FIGS. 1A and 1B). While PMK inplants resides in peroxisomes, it is assumed that bacterial RcMPDlocalizes to the cytoplasm. In this scenario, the increase in terpenoidproduction observed in RcMPD transgenic lines may result from theintroduction of an alternative biosynthetic route that bypasses theperoxisomal MVAP import and IPP export steps associated with thenaturally-occurring plant MVA pathway 100 a. To test this hypothesis,the in planta subcellular localization of the introduced RcMPD proteinwas examined.

GFP was fused to either the N-terminus or C-terminus of RcMPD andtransiently expressed in Nicotiana benthamiana leaves. Briefly, theRcMPD ORF was cloned into the binary vectors pK7FWG2 and pK7WGF2 withand without stop codons to generate N- and C-terminal GFP-fusionconstructs, respectively. mCherry markers for peroxisome (px-rk CD3-983)and mitochondria (mt-rk CD3-991) were co-infiltrated with GFP constructspursuant to methodologies known in the art. Constructs, markers and EVcontrols were transformed into Agrobacterium (EHA105) and infiltratedinto 3-week-old N. benthamiana leaves as previously described andpursuant to methods known in the art. Plant tissues were analyzed 1-2days after infiltration using the Nikon A1Rsi laser scanning confocalmicroscope.

As shown in FIG. 13, the GFP signal for the C-terminal GFP fusionprotein RcMDP-GFP was detected in the cytoplasm. In contrast andunexpectedly, the green fluorescence of GFP (labelled G) associated withN-terminal GFP fusion protein GFP-RcMPD was observed in peroxisomes (forreference, RFP is labelled P), which supports that the bacterial RcMPDpossesses a cryptic peroxisomal targeting signal that is blocked uponfusion of GFP to RcMPD's C-terminus. Further, examination of thebacterial RcMPD sequence (see FIG. 13g ) also revealed a peroxisomaltargeting signal type 2 (PTS2 motifs labeled in box 1301; (R/K) (L/V/I)X5 (H/Q) (L/A) labelled box 1302) which, despite not being present inthe N-terminus, was perhaps still recognized by the plant peroxisomalimport machinery. While initially assumed the increase in terpenoidformation was due to bypass of the peroxisome, the above construct datanegated that theory. Instead, it was determined that the RcMPD did infact localize in the peroxisome and that substrate and transport out ofperoxisomes are not limiting factors with respect to terpenoidsynthesis.

Given RcMPD's peroxisomal localization, the enhanced terpenoidproduction in RcMPD- and MPD4-AtIPK tobacco transgenics shown hereinindicates that generated IP can be transported out of this organelle(likely in a similar way as IPP). These results also support thatperoxisomal MVAP levels are sufficient to achieve the observed increasesin overall terpenoid production (see FIG. 11B). Therefore, flux throughthe classical MVA pathway 100 a is, at least in part, limited by theconversion of MVAP to MVAPP by PMK (a heretofore unsuspected regulatoryhub in the plant MVA pathway).

To further support this finding, Arabidopsis PMK was transientlyoverexpressed in wild-type tobacco leaves under control of the CaMV 35Spromoter using the methodologies previously described (see FIG. 14A) (N.tabacum PMK ORF's were codon-optimized for E. coli expression andsynthesized by SGI-DNA). As shown in FIG. 14B, relative to theempty-vector control, AtPMK overexpression led to 4- and 44-foldincreases in β-caryophyllene and 5-epi-aristolochene levels,respectively, with negligible effects on monoterpene levels. Next, tocatalyze the rate-limiting step of the MVA pathway 100 a, AtHMGR wastransiently coexpressed with AtPMK in wild-type tobacco leaves. For thisstudy, AtHMGR was also transiently overexpressed alone (as compared toEV controls), which led to increased emission of sesquiterpenes (2.8-and 9.4-fold for β-caryophyllene and 5-epi-aristolochene, respectively),but no discernable effect on monoterpene levels.

Compared to overexpressing AtPMK and AtHMGR individually, the resultsshown in FIG. 14B clearly illustrate that overexpressing the two genestogether further increased β-caryophyllene and 5-epi-aristolocheneemission by 17- and 63-fold, respectively, relative to the EV control.Coexpressing AtPMK and AtHMGR also increased emission of monoterpenes by4.6-fold (on average), which is indicative of sufficient IPP levelsbeing produced in the cytoplasm to drive plastidial terpenoidbiosynthesis.

These results demonstrate that PMK does indeed share control of fluxwith HMGR through the plant MVA pathway 100 a and may in fact pull onthe peroxisomal MVAP pool imported from the cytoplasm. This shift in thetransport equilibrium toward peroxisomes depends on PMK catalyticefficiency and PMK's peroxisomal concentration. In contrast to all otherenzymes of the classical MVA pathway 100 a except MKs, PMKs are encodedby single copy genes. Previous biochemical characterization ofArabidopis PMK (AtPMK, At1g31910) as well as N. tabacum PMK (NtPMK, XP016504246) described herein, shows that PMKs possess high specificityfor MVAP with K_(M) values of 12 μM¹³ and 31 μM, respectively.

TABLE 3 | Kinetic parameters of N. tabacum PMK and IPK. K_(M) k_(cat)k_(cat)/K_(M) Organism UniProt Enzyme Substrate (μm) (s⁻¹) (M⁻¹ s⁻¹) N.A0A1S4CT09 PMK (R)-MVAP 31 ± 7  22 ± 1  7.1 × 10⁵ ± 1.6 tabacumA0A1S4BSB2 IPK IP 4.0 ± 1.5 0.93 ± 0.05 2.3 × 10⁵ ± 0.9 DMAP 8.5 ± 2.31.20 ± 0.06 1.4 × 10⁵ ± 0.4 GP 100 ± 60  0.0017 ± 0.0002 1.7 × 10³ ± 1  

The catalytic efficiencies (k_(cat)/K_(M)) of PMKs from both specieswere comparable (1.7×10⁶ and 7.1×10⁵ M⁻¹s⁻¹ for AtPMK¹³ and NtPMK,respectively). These efficiencies are also similar to AtIPK (1.0×10⁵M⁻¹s⁻¹) characterized previously and NtIPK (XP_009616074) (2.3×10⁵M⁻¹s⁻¹) and in Table 3. As PMK is catalytically efficient, and itsencoding gene is one of the most highly expressed MVA pathway genes,unexplored transcriptional and/or posttranscriptional regulation maycontrol PMK activity and metabolic flux through the MVA pathway 100 a.

The unexpected, and heretofore unknown, peroxisomal localization of theoverexpressed RcMPD, which competes with the same MVAP substrate as PMK,demonstrates that the MVAPP formed by PMK is likely a limiting factorfor the biosynthesis of downstream MVA-derived terpenoids. Indeed,transient overexpression of AtPMK in wild-type tobacco led to someincrease in emitted sesquiterpenes, as did overexpression of AtHMGR(FIG. 11B); however, coexpression of AtPMK and AtHMGR substantiallyenhanced the formation of both sesquiterpenes and monoterpenes relativeto the EV controls. This supports that these two genes are positivelyepistatic and encode enzymes that have major roles in controlling fluxthrough the plant MVA pathway 100 a. When AtHMGR was coexpressed withRcMPD, the latter providing a bypass to the PMK-catalyzed reaction,again, synergistic effects were observed for both emitted sesquiterpenesand monoterpenes (FIG. 11B). These results further support PMK being anunsuspected regulatory hub in the plant MVA pathway 100 a and suggest arole for IP in regulating the formation of both MVA and MEPpathway-derived terpenoids.

All enzyme assays described in the present disclosure were performed asfollows. Phosphatase activity was monitored at 37° C. using BIOMOL Greenreagent (Enzo Life Sciences) to detect and quantify released phosphateat 623 nm using a phosphate standard curve. The optimum pH for activitywas determined using a three-component buffer system of 50 mM aceticacid, 50 mM IVIES, and 100 mM Tris-HCl with 0.1 mM IPP as substrate.Magnesium ion dependence was investigated with 1 mM to 20 mM MgCl₂.Steady-state parameters were determined in 100 mM TAPS pH 8.5 with 10 mMMgCl₂ and varied concentrations of substrates IPP, DMAPP, GPP, FPP, and8-oxo-dGTP (TriLink Biotechnologies). Assays with 8-oxo-dGTP included0.05 U of inorganic pyrophosphatase (ThermoFisher Scientific) per 0.1 mlassay. Reactions were initiated by the addition of enzyme and quenchedby the addition of BIOMOL Green.

Activities of the NtPMK and MIPK were analyzed by the lactatedehydrogenase/pyruvate kinase coupled assay as detailed in the IPKArticle. Briefly, assays were conducted in 100 mM NatHepes pH 7.5supplemented with 8 mM MgCl₂ and 100 mM KCl with coupling enzymespyruvate kinase/lactate dehydrogenase (Sigma), 4 mM ATP, 2 mMphosphoenolpyruvate, 0.16 mM NADH and varied concentrations of (R)-MVAP(Sigma) and IP (isoprenoids) as substrate for NtPMK and MIPK,respectively. Reactions were monitored at 340 nm and 30° C. Controlsincluded assays without enzyme and without substrate. All enzyme assayswere performed at an appropriate enzyme concentration so that thereaction velocity was linear and proportional to the enzymeconcentration during the incubation time period. Kinetic data was fittedusing Prism (GraphPad Software) to the Michaelis-Menten equation tocompute K_(M) and k_(cat). At least triplicate assays were performed forall data points.

Resulting Findings and Systems, Methods, and Platforms

The findings described herein can be leveraged to provide new strategiesfor high-level production of economically valuable terpenes andterpenoids. In at least one embodiment of the present disclosure, atransgenic plant comprises a first heterologous nucleic acid encoding apolypeptide having HMGR activity and a second heterologous nucleic acidencoding a second polypeptide. In at least one embodiment, the firstand/or second heterologous nucleic acid(s) may be from Arabidopsis. Thepolypeptide encoded by the second heterologous nucleic acid may comprisea polypeptide that introduces de novo formation of IP in the transgenicplant or a polypeptide having PMK activity and, in such cases, thesecond heterologous nucleic acid may comprise a bacterial gene such as,without limitation, an MPD gene from Roseiflexus castenholzii (Rc). Suchgenes may be transiently or stably overexpressed depending on thedesired application.

In at least one embodiment, one or both of the first and secondheterologous nucleic acids are operably linked to a regulatory elementfor directing expression of such heterologous nucleic acid. For example,the regulatory element may comprise a promoter. In at least oneembodiment, the promoter comprises a tissue-specific promoter to conferexpression of the first or second heterologous nucleic acid molecule tospecific plant tissue(s) or cells of the transgenic plant such as, forexample, a leaf, root, flower, developing ovule, seeds, fruits, embryo,meristems, etc.

Where the polypeptide encoded by the second heterologous nucleic acidhas MPD activity, the resulting transgenic plant exhibits increasedactivity of both HMGR and MPD and, when grown under the desiredconditions, exhibits an increase in the metabolically available IPwithin the transgenic plant cells as a result of the overexpression ofMPD. This provides a bypass to the PMK-catalyzed reaction and, in turn,results in a significant uptick in the production of endogenousterpenoids (at least a 20-100 fold increase) as compared to theterpenoid production capabilities of a wild-type plant or thosetransgenic plants prepared pursuant to conventional methodologies.Indeed, such coexpression of HMGR and MPD in the transgenic plant of thepresent disclosure can increase monoterpene formation by up to 20-foldand sesquiterpene production by up to or greater than 130-fold (both ascompared to the production of such terpenoids in wild-type plants).

At least one of the reasons for this greatly enhanced increase interpenoid production relates to the novel manipulation of both the MEPand MVA pathways. While the overexpression of HMGR manipulates thecytosolic MVA pathway 100 a by increasing the metabolically availableIP, overexpression of MPD as disclosed herein concurrently introduces denovo IP production in the transgenic plant. In comparison, transgenicplants of the prior art rely solely on the manipulation of theplastidial MEP pathway 102 and only see a marginal increase in terpenoidproduction of about 2-fold relative to wild-type.

Alternatively, where the second heterologous nucleic acid transgenicplant of the present disclosure has PMK activity such that both HMGR andPMK (Arabidopsis HMGR and/or PMK, for example) are overexpressed in thetransgenic plant, PMK's peroxisomal concentration increases thusincreasing flux through the classical MVA pathway 100 a and drivingplastidial terpenoid biosynthesis. As supported by the data of thepresent disclosure, the overexpression of HMGR and PMK together resultsin substantially enhanced formation of both sesquiterpenes andmonoterpenes relative to EV controls (i.e. wild-type plant cells); at ornear a 20-fold increase in production of endogenous monoterpenes and ator near a 130-fold increase in endogenous sesquiterpene production.

Notably, the transgenic plant of the present disclosure may also begenetically modified to produce exogenous terpenoids, the production ofwhich will also be amplified using the techniques of the presentdisclosure. These embodiments are particularly useful where a desiredterpenoid is not naturally produced by the wild-type plant. Accordingly,the transgenic plant of the present disclosure may further comprise athird heterologous nucleic acid comprising a sequence encoding asynthase for catalyzing the formation an exogenous terpene product ofinterest. Accordingly, in addition to producing increased amounts ofendogenous terpenes due to the coexpression of HMGR/MPD and/or HMGR/PMK,the novel transgenic plant will also produce the exogenous terpeneproduct of interest in amplified amounts as it utilizes the samebiosynthesis pathway and flux therethrough is increased. In this manner,the transgenic plants of the present disclosure not only provide for thesignificant amplification of endogenous terpenoid production, but alsothe ability to produce one or more exogenous terpenes of interest inhigher amounts than has been heretofore achieved using conventionalmethodologies.

Now referring to FIG. 15, methods for producing terpenoids using atransgenic plant are also provided in view of the disclosure providedherein. In at least one embodiment, such a method 1500 comprises a step1502 of providing a transgenic plant or plant cells co-expressing bothHMGR and MPD or PMK transgenes. For example, the transgenic plant/cellsmay comprise a first heterologous nucleic acid encoding a polypeptidehaving HMGR activity and a second heterologous nucleic acid encoding apolypeptide having MPD or PMK activity such that HMGR and thepolypeptide having MPD or PMK activity are stably or transientlyoverexpressed in the transgenic plant as compared to a correspondingwild-type plant.

Step 1502 may be performed according to the concepts and methodologiesdescribed herein and/or using methodologies now-known in the art orhereinafter developed for the genetic manipulation of plants and plantcells. For example, and without limitation, plant cells may betransformed with a transformation vector carrying the first heterologousnucleic acid encoding a polypeptide having HMGR activity to increaseHMGR enzyme content in the transgenic plant. By way of nonlimitingexample, such vector may contain Arabidopsis hmgr under the control of aCaMV 35S promoter.

Additionally or alternatively, the plant cells are cotransformed with abacterial MPD gene (from Roseiflexus castenholzii, for example) thatencodes an enzyme that possesses specificity for the efficientdecarboxylation of MVAP to IP and thereby introduces de novo IPformation in the transgenic plant. In at least one embodiment, the MPDgene may be stably or transiently overexpressed in the transgenic plantunder the control of a CaMV 35S promoter (or the like). This creates abifurcation in the canonical MVA pathway and allows for the productionof IP without dephosphorylating IPP (similar to the alternative MVApathway in some bacteria and archaea).

Still further, plant cells may be transformed with a vector carrying thesecond heterologous nucleic acid encoding a polypeptide having PMKactivity to increase PMK content in the transgenic plant. By way of anonlimiting example, such transformation vector may comprise anArabidopsis PMK vector under the control of a CaMV 35S promoter.

In at least one exemplary embodiment, the transgenic plant/cells furthercomprises a third heterologous nucleic acid comprising a sequenceencoding a synthase for catalyzing the formation of an exogenous terpeneproduct of interest. In such cases, at step 1503, the plant and/or plantcells are transformed with a vector carrying the third heterologousnucleic acid such that the resulting transgenic plant and/or cellsexpresses at least a portion of the exogenous terpene product ofinterest. For example, in at least one embodiment, the thirdheterologous nucleic acid encodes Santalum album, S. austrocaledonicum,or S. spicatum santalene synthase, which catalyzes the formation of amixture of sesquiterpenoids in the resulting transgenic plant.

While specific plant and bacterial species are listed herein by way ofexplanatory examples, such examples are not intended to be limiting.Indeed, the present disclosure should be broadly interpreted to includeany other species of genes that one of ordinary skill in the art maycontemplate or find beneficial in view of the findings of the presentdisclosure and the current state of the art.

After the transgenic plants or cells of step 1502, and optionally step1503, are obtained, explants are selected out that contain and coexpressboth the first and second heterologous nucleic acids and grown intomature plants that produce terpenoids. Where the method 1500 includesstep 1503 such that transgenic plant/cells also incorporate the thirdheterologous nucleic acid, the selection criteria at step 1504 furtherincludes those explants that also coexpress at least a portion of theexogenous terpene product of interest.

Perhaps more specifically, in at least one embodiment, following thetransformation step(s) 1502/1503 (as desired) of method 1500, aplurality of the resulting transgenic plant cells are cultured at step1504. A subset of transgenic plant cells that overexpress the genes ofinterest (e.g., HMGR+MPD and/or PMK and, optionally, a third exogenousterpene synthesis gene) are then selected and isolated from the culture.At step 1504 such transgenic plant or cells is/are grown under desiredconditions to achieve a mature transgenic plant that overexpresses HMGRand MPD or HMGR and PMK (and the exogenous terpenoid synthetase, ifdesired) as compared to a corresponding wild-type plant. Pursuant to theexperimental data set forth herein, the coexpression of HMGR and MPD orPMK affects flux toward downstream terpenoid production and, thus,results in between about at least a 20-100 fold increase of terpenoidproduction as compared to a wild-type plant (and, up to or greater thana 130-fold increase of sesquiterpene production).

If desired, one or more terpenoids (endogenous or exogenous) may also beisolated and/or harvested from the transgenic plant at optional step1506 pursuant to methodologies commonly known in the art. Additionallyor alternatively, because terpenoid production in plants (e.g., crops)enhances the natural defenses of the plant, method 1500 may be leveragedas a pest and/or pathogen-management application or strategy. In suchembodiments, the method 1500 further comprises optional step 1508wherein crops comprising one or more of the transgenic plants of thepresent disclosure are grown in a plant growing area pursuant to methodscommonly known in the art. Such crops may, in at least one embodiment,be agricultural. Furthermore, the crops may comprise seasonal orperennial plants and/or the plant growing area may comprise an outdoorfield or other growing area (whether in a greenhouse or other indoorfacility).

Accordingly, unlike conventional techniques, the transgenic plants andmethods of the present disclosure provide for a significant increase interpenoid production. While various embodiments of transgenic plants,platforms, and methods hereof have been described in considerabledetail, the embodiments are merely offered by way of non-limitingexamples. Many variations and modifications of the embodiments describedherein will be apparent to one of ordinary skill in the art in light ofthe disclosure. It will therefore be understood by those skilled in theart that various changes and modifications may be made, and equivalentsmay be substituted for elements thereof, without departing from thescope of the disclosure. Indeed, this disclosure is not intended to beexhaustive or too limiting. The scope of the disclosure is to he definedby the appended claims, and by their equivalents.

Further, in describing representative embodiments, the disclosure mayhave presented a method and/or process as a particular sequence ofsteps. However, to the extent that the method or process does not relyon the particular order of steps set forth herein, the method or processshould not be limited to the particular sequence of steps described. Asone of ordinary skill in the art would appreciate, other sequences ofsteps may be possible. Therefore, the particular order of the stepsdisclosed herein should not be construed as limitations on the claims.In addition, the claims directed to a method and/or process should notbe limited to the performance of their steps in the order written, andone skilled in the art can readily appreciate that the sequences may bevaried and still remain within the spirit and scope of the presentdisclosure.

It is therefore intended that this description and the appended claimswill encompass, all modifications and changes apparent to those ofordinary skill in the art based on this disclosure.

1. A transgenic plant comprising a first heterologous nucleic acidencoding a polypeptide having 3-hydroxy-3-methylglutarylCoA reductase(HMGR) activity and a second heterologous nucleic acid encoding apolypeptide that introduces de novo formation of isopentenyl phosphate(IP) in the transgenic plant or a polypeptide having phosphomevalonatekinase (PMK) activity, wherein the polypeptide having HMGR activity andthe polypeptide encoded by the second heterologous nucleic acid areoverexpressed in the transgenic plant as compared to a correspondingwild-type plant.
 2. The transgenic plant of claim 1, wherein the firstheterologous nucleic acid encoding a polypeptide having HMGR activity isfrom Arabidopsis.
 3. The transgenic plant of claim 1, wherein thepolypeptide encoded by the second heterologous nucleic acid introducesde novo formation of IP in the transgenic plant and hasphosphomevalonate decarboxylase (MPD) activity.
 4. The transgenic plantof claim 3, wherein the second heterologous nucleic acid comprises abacterial gene.
 5. The transgenic plant of claim 3, wherein thetransgenic plant produces an increased amount of metabolically availableIP relative to an amount of metabolically available IP produced in acorresponding wild-type plant.
 6. The transgenic plant of claim 1,further comprising a third heterologous nucleic acid comprising asequence encoding a synthase for catalyzing the formation of anexogenous terpenoid product of interest, wherein the transgenic plantexpresses at least a portion of the exogenous terpenoid product ofinterest.
 7. The transgenic plant of claim 1, wherein polypeptideencoded by the second heterologous nucleic acid has PMK activity andmonoterpene and sesquiterpene production of the transgenic plant is ator near 20-fold greater and at or near 130-fold greater, respectively,than monoterpene and sesquiterpene production in the correspondingwild-type plant.
 8. The transgenic plant of claim 1, wherein one or bothof the first and second heterologous nucleic acids is operably linked toa regulatory element for directing expression of the first and secondheterologous nucleic acids.
 9. The transgenic plant of claim 8, whereinthe regulatory element comprises a tissue-specific promoter fordirecting expression of the first or second heterologous nucleic acid inthe plant cells of a leaf, root, flower, developing ovule or seed of thetransgenic plant.
 10. The transgenic plant of claim 1, wherein thetransgenic plant is selected from the group consisting of: tobacco,rice, flax, wheat, barley, rye, corn, potato, pea, lettuce, cabbage,cauliflower, broccoli, turnip, radish, spinach, asparagus, onion,pepper, celery, squash, pumpkin, cucumber, strawberry, grape, raspberry,blackberry, pineapple, avocado, mango, banana, soybean, tomato, sorghum,sugarcane, and algae.
 11. A method for producing terpenoids using atransgenic plant comprising the steps of: providing a transgenic plantcomprising a first heterologous nucleic acid encoding a polypeptidehaving HMGR activity and a second heterologous nucleic acid encoding apolypeptide having MPD or PMK activity such that the first and secondheterologous nucleic acids are overexpressed in the transgenic plant ascompared to a corresponding wild-type plant; and growing the transgenicplant under desired conditions such that one or more terpenoids ofinterest are produced.
 12. The method of claim 11, further comprisingthe step of isolating one or more terpenoids of interest from thetransgenic plant after growth.
 13. The method of claim 11, wherein thetransgenic plant is grown in the presence of labeled carbon dioxide,water, or a combination thereof.
 14. The method of claim 11, wherein thestep of providing a transgenic plant further comprises the steps of:transforming plant cells with Agrobacterium containing a vector carryingthe first heterologous nucleic acid encoding a polypeptide having HMGRactivity in a context that allows for the overexpression of the firstheterologous nucleic acid in a transgenic plant; transforming the plantcells with a gene containing a vector carrying the second heterologousnucleic acid encoding a polypeptide having PMK activity in a contextthat allows for the overexpression of the second heterologous nucleicacid in a transgenic plant; selecting transformants that overexpressboth the first and second heterologous nucleic acids; and growing thetransformants into a transgenic plant.
 15. The method of claim 11,further comprising the step of transforming plant cells with a nucleicacid sequence operably linked to one or more regulatory elements fordirecting expression of the nucleic acid sequence in the plant cells,the nucleic acid sequence encoding a synthase for catalyzing theformation an exogenous terpenoid product of interest; and wherein thestep of selecting transformants that contain and overexpress both thefirst and second heterologous nucleic acids further comprises selectingtransformants that express at least a portion of the exogenous terpenoidproduct of interest.
 16. The method of claim 11, wherein the transgenicplant produces one or more terpenoids of interest at or over a 20-foldincrease relative to terpenoid production of each terpenoid of interestin a corresponding wild-type plant.
 17. A method for producing atransgenic plant cell culture comprising the steps of: obtainingtransgenic plant cells by co-transforming plant cells with: a firstheterologous nucleic acid encoding a polypeptide having HMGR activity,and a second heterologous nucleic acid encoding a polypeptide having PMKactivity; culturing a plurality of the transgenic plant cells; andselecting and isolating from the plurality of transgenic plant cells asubset of transgenic plant cells where expression of the first andsecond nucleic acids are amplified as compared to wild-type resulting ina transgenic plant cell culture.
 18. The method of claim 17, wherein:the step of obtaining transgenic plant cells further comprisesco-transforming the plant cells with a third nucleic acid comprising asequence encoding a synthase for catalyzing the formation an exogenousterpenoid product of interest; the method further comprises the step ofcultivating the transgenic plant cells so that at least a portion of theexogenous terpenoid product of interest encoded by the third nucleicacid is expressed by the transgenic cells; and the subset of transgenicplant cells in the selecting and isolating step further comprisestransgenic plant cells expressing at least a portion of the exogenousterpenoid product of interest.
 19. The method of claim 17, wherein thetransgenic plant cell culture produces β-caryophyllene and5-epi-aristolochene at or near a 17-fold and a 63-fold increase,respectively, relative to β-caryophyllene and 5-epi-aristolocheneemission in a corresponding wild-type plant cell culture.
 20. The methodof claim 17, further comprising the step of growing transgenic planttissue from the subset of transgenic plant cells.